Protocol to Identify GFP Transgenic Mouse
Protocol to Identify GFP Transgenic Mouse
Method to Isolate DNA from Ear Notch:
Ear Notch Buffer (80 mL):
0.298g KCl + 70 mL Sterile H2O
533 mL of 1.5M TrisCl (pH 8.0)
100mL of 2M MgCl2
360 mL of IGEPAL
360mL of Tween 20
8.647 mL Sterile H2O
Method to Isolate DNA:
1. Take 500mL of ear notch buffer and add 25mL of Proteinase K.
2. Aliquot 40mL of buffer with Proteinase K into each Epperdorf tube.
3. Take an ear notch sample from one ear of mouse.
4. Digest 3 to 4 hours @ 56 to 65oC. Mix occasionally.
5. Take out DNA for PCR and freeze sample immediately!!!
**** Keep this DNA on ice or frozen at all times as it is will quickly degrade at room temperature!!! ****
SD-PCR Protocol:
PCR Tube Contents / Volume (Total = 50mL)Sterile H2O / 32.5mL
10X Taq Buffer / 5mL
5mM dNTPs / 2mL
4mM Primer #1 (Fwd.) / 4mL
4mM Primer #2 (Rev.) / 4mL
DNA Template / 2mL
Taq Polymerase (add at SD-PCR step 2) / 0.5mL
Note: Do not substitute Vent for Taq as Vent will not work!
Primer #1 (Fwd.) Sequence: 5’ TGG TGA TGT TAA TGG GCA CAA 3’
Primer #2 (Rev.) Sequence: 5’ CAG CAC GTG TCT TGT AGT TCC CG 3’
SD-PCR:
1. 94oC for 10 min. (to destroy Proteinase K)
2. 80oC for 30 sec. During this time period PAUSE PCR MACHINE and add 0.5 mL Taq polymerase to each tube.
3. 3 cycles at: (94oC for 1 min., 69oC for 2 min., 72oC for 1.5 min.)
4. 3 cycles at: (94oC for 1 min., 66oC for 2 min., 72oC for 1.5 min.)
5. 3 cycles at: (94oC for 1 min., 63oC for 2 min., 72oC for 1.5 min.)
6. 3 cycles at: (94oC for 1 min., 60oC for 2 min., 72oC for 1.5 min.)
7. 3 cycles at: (94oC for 1 min., 57oC for 2 min., 72oC for 1.5 min.)
8. 13 cycles at: (94oC for 1 min., 55oC for 2 min., 72oC for 1.5 min.)
9. 72oC for 7 minutes.
10. Use a 1% agarose gel to analyze the PCR product. PCR product is ~ 300 bp.
Sample gel showing desired bands indicated (arrow):
Lane 1: 1Kb Plus DNA ladder; Lane 5: (-) Control; Lane 6: (+) Control; Lane 7: (+ GFP Transgenic Mouse) Control; Lanes 12 & 13: PCR product of DNA isolated from ear notches.